Method for detecting escherichia coli

ABSTRACT

Described herein are methods of detecting an infection and for detecting the presence or absence of microorganisms, for example, wound pathogens in a sample, by contacting a sample with an enzyme produced and/or secreted by the bacteria, and detecting modification or the absence of modification of the substrate, as an indicator of the presence or absence of the enzyme in the sample. The present invention also features a biosensor for detecting the presence or absence of bacteria in a sample.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/444,523, filed on Jan. 31, 2003. The entire teachings of the aboveapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Infections are a major source of healthcare expenditure. Approximately5% of all surgical wounds become infected with microorganisms, and thatfigure is considerably higher (10-20%) for patients undergoing abdominalsurgery. Bacterial species, such as Escherichia coli (E. coli) are themost frequently isolated organisms from infected wounds. Bacterialcolonization rates are significantly higher in the hospital setting,both among healthcare workers, and among patients. Moreover, thecolonizing organisms in the hospital environment are likely to beresistant to many forms of antimicrobial therapy, due to the strongselective pressure that exists in the nosocomial environment, whereantibiotics are frequently used. Most strains of Escherichia coli canharmlessly coexist with humans, for example, in their intestines, andare not likely to cause disease under normal circumstances. Somestrains, however, produce toxins that can cause severe, even lifethreatening disorders, including intestinal disorders, kidney disorders,and urinary tract infections.

Escherichia coli are one type of pathogenic microorganism that can befound in infections in the human body; others include, but are notlimited to Streptococcus pyogenes, Pseudomonas aeruginosa, Enterococcusfaecalis, Proteus mirabilis, Serratia marcescens, Enterobacter clocae,Acetinobacter anitratus, Klebsiella pneumoniae, and Staphylococcusspecies.

Infection, including wound infection due to any of the above organismsis a significant concern of hospitals. The most common way of preventingsuch infection is to administer prophylactic antibiotic drugs. Whilegenerally effective, this strategy has the unintended effect of breedingresistant strains of bacteria. The routine use of prophylacticantibiotics should be discouraged for the very reason that it encouragesthe growth of resistant strains.

Rather than using routine prophylaxis, a better approach is to practicegood anti-microbial management, i.e., keep area at risk for becominginfected away from bacteria before, during, and after surgery, andcarefully monitor the wound site or patient fluid for infection. Normalmonitoring methods include close observation of the wound site for slowhealing, signs of inflammation and pus, as well as measuring thepatient's temperature for signs of fever and testing the patient'sfluids, for example, urine, for signs of infection. Unfortunately, manysymptoms are only evident after the infection is already established.Furthermore, after a patient is discharged from the hospital they becomeresponsible for monitoring their own healthcare, and the symptoms ofinfection may not be evident to the unskilled patient.

A system or biosensor that can detect the early stages of infectionbefore symptoms develop would be advantageous to both patients andhealthcare workers. If a patient can accurately monitor the condition ofa wound after discharge, then appropriate antimicrobial therapy can beinitiated early enough to prevent a more serious infection.

SUMMARY OF THE INVENTION

It has been found that molecules, for example, proteins secreted bymicroorganisms, such as bacteria or fungi, expressed on the cell surfaceof microorganisms, or expressed on the surface of a cell infected with avirus or prion can serve as markers for the detection of the presence orabsence of the microorganism in a sample, for example, a wound or bodyfluid. Accordingly, the present invention features a method of detectingthe presence or absence of a microorganism, for example, E. coli in asample by detecting the presence or absence of a molecular marker forthe microorganism in the sample. In particular, the molecular markers tobe detected include proteins, such as enzymes that are specific to aspecies of microorganism.

In one aspect, the invention features a method for detecting thepresence or absence of a microorganism, for example, E. coli in asample, comprising the steps of contacting the sample with a detectablylabeled substrate for an enzyme produced and/or secreted by themicroorganism, under conditions that result in modification of thesubstrate by the enzyme; and detecting the modification or the absenceof the modification of the substrate. Modification of the substrateindicates the presence of the microorganism in the sample, and theabsence of modification of the substrate indicates the absence of themicroorganism in the sample. In particular, the substrate can consist oflabeled peptide that is cleaved by a protease enzyme to give a signalthat can be detected. Furthermore, this peptide can be designed with aparticular sequence of amino acid residues extending from one end of theoriginal substrate peptide as a “tag” for use in covalently coupling thesubstrate to a surface.

In another aspect, the present invention features a method fordiagnosing the presence or absence of an infection in a subject,comprising the steps of a) contacting a sample obtained from a wound ina subject with a detectably labeled substrate for an enzyme producedand/or secreted by a microorganism, for example, E. coli, underconditions that result in modification of the substrate by the enzyme;and b) detecting a modification or the absence of a modification of thesubstrate. Modification of the substrate indicates the presence of aninfection in the subject, and the absence of modification of thesubstrate indicates the absence of an infection in the subject.

In yet another aspect, the present invention features a method fordiagnosing the presence or absence of a wound infection in a subject,comprising the steps of a) contacting a subject with a detectablylabeled substrate for an enzyme produced and/or secreted by amicroorganism, for example, E. coli, under conditions that result inmodification of the substrate by the enzyme; and b) detecting amodification or the absence of a modification of the substrate.Modification of the substrate indicates the presence of a woundinfection in the subject, and the absence of modification of thesubstrate indicates the absence of a wound infection in the subject.

In another aspect, the invention features a biosensor for detecting thepresence or absence of a microorganism, for example, E. coli, comprisinga solid support and a detectably labeled substrate for an enzymeproduced and/or secreted by the microorganism, wherein the substrate isattached to the solid support.

In still another aspect, the present invention features a kit fordetecting an infection, comprising a biosensor for detecting thepresence or absence of a microorganism in a sample, and one or morereagents for detecting the presence of the microorganism that is thecausative agent of the infection. For example, the reagent can be usedto detect an enzyme secreted by the microorganism. In particular, thereagent can be used to detect the modification of the substrate of thebiosensor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the cleavage of target substrate ecot1 (T1)(relative fluorescence) in samples containing various bacteria, asindicated. All bacterial samples are directly from culture and includecells and media. (Legend abbreviations: Buffer=20 mM tris buffer (pH7.4) with 150 mM NaCl, Peptide T1=labeled peptide substrate)

FIG. 2 is a graph of the cleavage of target substrate ecot2 (T2)(relative fluorescence) in samples containing various bacteria, asindicated. All bacterial samples are directly from culture and includecells and media. (Legend abbreviations: Buffer=20 mM tris buffer (pH7.4) with 150 mM NaCl, Peptide T2=labeled peptide substrate)

FIG. 3 is a graph of the cleavage of target substrate ecot2 (T2)(relative fluorescence) in samples containing various bacteria, asindicated, plus fetal bovine serum (FBS). All bacterial samples aredirectly from culture and include cells and media. (Legendabbreviations: Buffer=20 mM tris buffer (pH 7.4) with 150 mM NaCl,Peptide T2=labeled peptide substrate, FBS=fetal bovine serum)

FIG. 4 is a graph of the cleavage of target substrate ecot2 (T2)(relative fluorescence) in simulated wound fluid samples containingvarious bacteria plus bovine serum albumin (BSA). All bacterial samplesare directly from culture and include cells and media. (Legendabbreviations: Buffer=20 mM tris buffer (pH 7.4) with 150 mM NaCl,Peptide T2=labeled peptide substrate)

FIG. 5 is a graph of cleavage of protease substrate T3 (relativefluorescence) over time in samples containing buffer, buffer plus T3,buffer plus T3C (crude peptide), or culture including cells and mediafrom Pseudomonas, E. coli, S. aureus (Staph aureus), S. epidermidis(Staph epidermidis), S. Salivarius (Strep salivarius), S. pyogenes(Strep pyogenes), Enterococcus, or Serratia.

DETAILED DESCRIPTION OF THE INVENTION

As part of their normal growth processes, many microorganisms secrete anumber of enzymes into their growth environment. These enzymes havenumerous functions including, but not limited to, the release ofnutrients, protection against host defenses, cell envelope synthesis (inbacteria) and/or maintenance, and others as yet undetermined. Manymicroorganisms also produce enzymes on their cell surface that areexposed to (and interact with) the extracellular environment. Many ofthese enzymes are specific to the microorganism that secretes them, andas such, can serve as specific markers for the presence of thosemicroorganisms. A system that can detect the presence of these enzymesthat are produced and/or secreted can equally serve to indicate thepresence of the producing/secreting microorganism. Alternatively, asystem that can detect the absence of these enzymes that are producedand/or secreted can equally serve to indicate the absence of theproducing/secreting microorganism. Such a detection system is useful fordetecting or diagnosing an infection. As used herein, an “infection”means a disorder caused by exposure to a pathogenic microorganism. Inone example, the microorganism is E. coli. In another example, thedisorder is a wound infection, an intestinal disorder, food poisoning, akidney disorder, or a urinary tract infection.

A microorganism detection test system, as described herein can betailored to detect one specific microorganism, for example, E. coli byidentifying a protein such as a secreted enzyme specific to themicroorganism to be detected. Alternatively, a test system can bedesigned to simultaneously identify more than one microorganism species(for example, at least 2, at least 5, or at least 10 differentmicroorganism species), such as those that commonly cause infections.Identifying those enzymes that are common to certain classes ofpathogenic microorganisms, but which are not present in non-pathogenicmicroorganisms is one way to achieve this goal. Such enzymes can beidentified, for example, with a computer based bioinformatics screen ofthe microbial genomic databases. By using enzymes as the basis fordetection systems, sensitive tests can be designed, since even a verysmall amount of enzyme can catalyze the turnover of a substantial amountof substrate.

The present invention pertains to the identification of bacterialproteins that are specific for microorganisms that are the causativeagent of an infection. The presence of a pathogenic bacterium can bedetected by designing a synthetic substrate that will specifically reactwith an enzyme that is present on the surface of the cell or secreted.These synthetic substrates can be labeled with a detectable label suchthat under conditions wherein their respective enzymes specificallyreact with them, they undergo a modification, for example, a visiblecolor change that is observed.

The enzymes that are used in the bacteria detection method of thepresent invention are preferably infection-specific enzymes. As usedherein, an infection-specific enzyme is an enzyme produced and/orsecreted by a pathogenic bacteria, but is not produced and/or secretedby a non-pathogenic bacteria. Examples of pathogenic bacteria include,but are not limited to staphylococcus (for example, Staphylococcusaureus, Staphylococcus epidermidis, or Staphylococcus saprophyticus),streptococcus (for example, Streptococcus pyogenes, Streptococcuspneumoniae, or Streptococcus agalactiae), enterococcus (for example,Enterococcus faecalis, or Enterococcus faecium), corynebacteria species(for example, Corynebacterium diptheriae), bacillus (for example,Bacillus anthracis), listeria (for example, Listeria monocytogenes),Clostridium species (for example, Clostridium perfringens, Clostridiumtetanus, Clostridium botulinum, Clostridium difficile), Neisseriaspecies (for example, Neisseria meningitidis, or Neisseria gonorrhoeae),E. coli, Shigella species, Salmonella species, Yersinia species (forexample, Yersinia pestis, Yersinia pseudotuberculosis, or Yersiniaenterocolitica), Vibrio cholerae, Campylobacter species (for example,Campylobacter jejuni or Campylobacter fetus), Helicobacter pylori,pseudomonas (for example, Pseudomonas aeruginosa or Pseudomonas mallei),Haemophilus influenzae, Bordetella pertussis, Mycoplasma pneumoniae,Ureaplasma urealyticum, Legionella pneumophila, Treponema pallidum,Leptospira interrogans, Borrelia burgdorferi, mycobacteria (for example,Mycobacterium tuberculosis), Mycobacterium leprae, Actinomyces species,Nocardia species, chlamydia (for example, Chlamydia psittaci, Chlamydiatrachomatis, or Chlamydia pneumoniae), Rickettsia (for example,Rickettsia ricketsii, Rickettsia prowazekii or Rickettsia akari),brucella (for example, Brucella abortus, Brucella melitensis, orBrucella suis), Proteus mirabilis, Serratia marcescens, Enterobacterclocae, Acetinobacter anitratus, Klebsiella pneumoniae and Francisellatularensis. Preferably, the infection-specific bacteria isstaphylococcus, streptococcus, enterococcus, bacillus, Clostridiumspecies, E. coli, yersinia, pseudomonas, Proteus mirabilis, Serratiamarcescens, Enterobacter clocae, Acetinobacter anitratus, Klebsiellapneumoniae or Mycobacterium leprae. For example, the infection-specificenzyme can be produced and/or secreted by Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus pyogenes, Pseudomonasaeruginosa, Enterococcus faecalis, Proteus mirabilis, Serratiamarcescens, Enterobacter clocae, Acetinobacter anitratus, Klebsiellapneumoniae and/or Escherichia coli.

Preferably, the enzyme is one or more of the following: phospholipase Aprotein, outer membrane protein T (ompT), or other omp proteins. Thesequences of these proteins can be obtained by carrying out searches onprotein sequence databases, for example, GenBank, and one skilled in theart can carry out such a search. Gene encoding such proteins can also becloned using cloning techniques known to one of skill in the art.

Substrates for use in the present invention include any molecule, eithersynthetic or naturally-occurring that can interact with an enzyme of thepresent invention. Examples of substrates include those substratesdescribed herein, as well as substrates for these enzymes that are knownin the art. Other examples of substrates include ecot1 (T1) derivedfluorescent peptides, for example, Edans-DSRPVRRRRRPRVSK-Dabcyl (SEQ IDNO: 1) or ecot2 (T2) derived fluorescent peptides, for example,Edans-KVSRRRRRGGD-Dabcyl (SEQ ID NO: 2), which can be cleaved by theompT protein of pathogenic E. coli. Such substrates described herein canbe obtained from commercial sources, e.g., Sigma (St. Louis, Mo.), orcan be produced, e.g., isolated or purified, or synthesized usingmethods known to those of skill in the art.

The enzymes of the present invention can modify substrates, for example,proteins or polypeptides by cleavage, and such modification can bedetected to determine the presence or absence of a pathogen in a sample.One method for detecting modification of a substrate by an enzyme is tolabel the substrate with two different dyes, where one serves to quenchthe fluorescence of the other dye by fluorescence resonance energytransfer (FRET) when the molecules, for example, dyes or colorimetricsubstances are in close proximity, and is measured by detecting changesin fluorescence.

FRET is the process of a distance dependent excited state interaction inwhich the emission of one fluorescent molecule is coupled to theexcitation of another. A typical acceptor and donor pair for resonanceenergy transfer consists of 4-[[-(dimethylamino) phenyl]azo]benzoic acid(Dabcyl) and 5-[(2-aminoethylamino]naphthalene sulfonic acid (Edans).Edans is excited by illumination with 336 nm light, and emits a photonwith wavelength 490 nm. If a Dabcyl moiety is located within 20angstroms of the Edans, this photon will be efficiently absorbed. Dabcyland Edans will be attached to opposite ends of a peptide substrate. Ifthe substrate is intact, FRET will be very efficient. If the peptide hasbeen cleaved by an enzyme, the two dyes will no longer be in closeproximity and FRET will be inefficient. The cleavage reaction can befollowed by observing either a decrease in Dabcyl fluorescence or anincrease in Edans fluorescence (loss of quenching).

If the substrate to be modified is a protein, peptide, or polypeptide,the substrate can be produced using standard recombinant proteintechniques (see for example, Ausubel et al., “Current Protocols inMolecular Biology,” John Wiley & Sons, (1998), the entire teachings ofwhich are incorporated by reference herein). In addition, the enzymes ofthe present invention can also be generated using recombinanttechniques. Through an ample supply of enzyme or its substrate, theexact site of modification can be determined, and a more specificsubstrate of the enzyme can be defined, if so desired. This substratecan also be used to assay for the presence of the pathogenic bacteria.

The substrates are labeled with a detectable label that is used tomonitor interactions between the enzyme and the substrate and detect anysubstrate modifications, for example, cleavage of the substrate or labelresulting from such interactions. Examples of detectable labels includevarious dyes that can be incorporated into substrates, for example,those described herein, spin labels, antigen or epitope tags, haptens,enzyme labels, prosthetic groups, fluorescent materials,chemiluminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzyme labels include horseradishperoxidase, alkaline phosphatase, β-galactosidase, andacetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride and phycoerythrin; an example of a chemiluminescent materialincludes luminol; examples of bioluminescent materials includeluciferase, luciferin, and aequorin, and examples of suitableradioactive material include ¹²⁵I, ¹³¹I, ³⁵S, and ³H. Other examples ofdetectable labels include Bodipy, Pyrene, Texas Red, Edans, DansylAziridine, IATR and fluorescein. Succimidyl esters, isothiocyanates, andiodoacetamides of these labels are also commercially available. Whendetectable labels are not employed, enzymatic activity can be determinedby other suitable methods, for example, detection of substrate cleavagethrough electrophoretic analysis, or other methods known to one skilledin the art.

One example of a preferred detectable label is a chromogenic dye thatallows monitoring of the hydrolysis of the substrate by themicroorganism. An example of such a dye is para-nitrophenol. Whenconjugated to a substrate molecule, this dye will remain colorless untilthe substrate is modified by the secreted enzyme, at which point itturns yellow. The progress of the enzyme-substrate interaction can bemonitored by measuring absorbance at 415 nm in a spectrophotometer.Other dyes that produce detectable modification, e.g., a visible colorchange, are known to those of skill in the art.

The sample in which the presence or absence of a bacteria, such as E.coli is detected, or an infection is diagnosed, can be, for example, awound, a body fluid, such as blood, urine, sputum, or wound fluid (forexample, pus produced at a wound site). The sample can also be anyarticle that bacteria may be contained on/in, for example, a wounddressing, a catheter, a urine collection bag, a blood collection bag, aplasma collection bag, a disk, a scope, a filter, a lens, foam, cloth,paper, a suture, swab, test tube, a well of a microplate, contact lenssolutions, food packaging material, or a swab from an area of a room orbuilding, for example, an examination room or operating room of ahealthcare facility, a bathroom, a kitchen, or a process ormanufacturing facility.

The present invention also features a biosensor for detecting a (one ormore, for example, at least 2, at least 5, at least 10, at least 20, atleast 30, at least 50, at least 75, or at least 100) marker proteinenzyme(s) described herein and for notifying a consumer of the presenceof the marker protein. A biosensor for use in healthcare settings orhome-use to detect infections comprising a (one or more) specificsubstrate(s) that is coupled to a solid support that is proximal to awound or other body fluid that is being monitored for bacterialcontamination is provided. Preferably, the substrate is covalently boundto a label and thus has a detection signal that upon proteolysis of thesubstrate-label bond indicates the presence of the bacteria. Such abiosensor can also be used in food preparation settings to detect forproducts that are contaminated with bacteria.

The biosensor is made by first determining the specific substrate of aspecific enzyme characteristic of the bacteria to be detected. Thedetermined specific substrate is labeled with one or more, andpreferably, a plurality of detectable labels, for example, chromatogenicor fluorescent leaving groups. Most preferably, the labeling groupprovides a latent signal that is activated only when the signal isproteolytically detached from the substrate. Chromatogenic leavinggroups include, for example, para-nitroanalide groups. Should thesubstrate come into contact with an enzyme secreted into a wound orother body fluid by bacteria or presented on the surface of a bacterialcell, the enzyme modifies the substrates in a manner that results indetection of such a modification, for example, a change in absorbance,which can be detected visually as a change in color (for example, on thesolid support, such as a wound dressing), or using spectrophotometrictechniques standard in the art.

The biosensor of the present invention also can comprise one or moresubstrates (for example, at least 2, at least 5, at least 10, at least20, at least 30, at least 50, at least 75, or at least 100 substrates)for produced and/or secreted enzymes of pathogenic bacteria. Thebiosensor is a solid support, for example, a wound dressing (such as abandage, or gauze), any material that needs to be sterile or free ofmicrobial contamination, for example, a polymer, disk, scope, filter,lens, foam, cloth, paper, or sutures, or an article that contains orcollects the sample (such as a urine collection bag, blood or plasmacollection bag, test tube, catheter, swab, or well of a microplate).

Typically, the solid support is made from materials suitable forsterilization if the support directly contacts the wound or infectedarea or sample. In one embodiment of the present invention, thebiosensor can be directly contacted with the wound or infected area. Insome instances, a sterile covering or layer is used to preventcontamination of the wound or body fluid upon such direct contact. Ifsuch sterile coverings are used, they will have properties that makethem suitable for sterilization, yet do not interfere with theenzyme/substrate interaction. Preferably, the portion of the biosensorthat comes into contact with the wound is also nonadherent to permiteasy removal of the biosensor from the sample surface. For example, ifthe biosensor comprises a wound dressing, the dressing contacts thewound for a time sufficient for the enzyme substrate to react and thenthe dressing is removed from the wound without causing further damage tothe wound or surrounding tissue.

Substrates suitably labeled with detectable labels, for example, achromogenic dye, and attached or incorporated into a sensor apparatus,can act as indicators of the presence or absence of pathogenic bacteriathat secrete the aforementioned enzymes. When more than one substrate isutilized, each may be labeled so as to distinguish it from another (forexample, using different detectable labels) and/or each may be localizedin a particular region on the solid support.

Substrates with hydrophobic leaving groups can be non-covalently boundto hydrophobic surfaces. Alternatively hydrophilic or hydrophobicsubstrates can be coupled to surfaces by disulfide or primary amine,carboxyl or hydroxyl groups. Methods for coupling substrates to a solidsupport are known in the art. For example, fluorescent and chromogenicsubstrates can be coupled to solid substrates using non-essentialreactive termini such as free amines, carboxylic acids or SH groups thatdo not effect the reaction with the pathogens. Free amines can becoupled to carboxyl groups on the substrate using, for example, a 10fold molar excess of eitherN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) orN-cyclohexyl-N′-2-(4′-methyl-morpholinium) ethyl carbodiimide-p-toluenesulphonate (CMC) for 2 hrs at 4° C. in distilled water adjusted to pH4.5 to stimulate the condensation reaction to form a peptide linkage. SHgroups can be reduced with DTT or TCEP and then coupled to a free aminogroup on a surface with N-e-Maleimidocaproic acid (EMCA, Griffith etal., FEBS Lett. 134:261-263, 1981). Example of substrates are providedherein.

The polypeptides of the invention also encompass fragments and sequencevariants of the polypeptide substrates described herein. Variantsinclude a substantially homologous polypeptide encoded by the samegenetic locus in an organism, i.e., an allelic variant, as well as othervariants. Variants also encompass polypeptides derived from othergenetic loci in an organism, but having substantial homology to apolypeptide substrate described herein Variants also includepolypeptides substantially homologous or identical to these polypeptidesbut derived from another organism, i.e., an ortholog. Variants alsoinclude polypeptides that are substantially homologous or identical tothese polypeptides that are produced by chemical synthesis. Variantsalso include polypeptides that are substantially homologous or identicalto these polypeptides that are produced by recombinant methods.

The percent identity of two amino acid sequences can be determined byaligning the sequences for optimal comparison purposes (e.g., gaps canbe introduced in the sequence of a first sequence). The amino acids atcorresponding positions are then compared, and the percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=# of identicalpositions/total # of positions×100). In certain embodiments, the lengthof the amino acid sequence aligned for comparison purposes is at least30%, preferably, at least 40%, more preferably, at least 60%, and evenmore preferably, at least 70%, 80%, 90%, or 100% of the length of thereference sequence. The actual comparison of the two sequences can beaccomplished by well-known methods, for example, using a mathematicalalgorithm. A preferred, non-limiting example of such a mathematicalalgorithm is described in Karlin et al., Proc. Natl. Acad. Sci. USA,90:5873-5877, 1993). Such an algorithm is incorporated into the BLASTprograms (version 2.2) as described in Schaffer et al. (Nucleic AcidsRes., 29:2994-3005, 2001). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs can be used.In one embodiment, the database searched is a non-redundant (NR)database, and parameters for sequence comparison can be set at: nofilters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; andGap Costs have an Existence of 11 and an Extension of 1.

In another embodiment, the percent identity between two amino acidsequences can be accomplished using the GAP program in the GCG softwarepackage (Accelrys Inc., San Diego, Calif.) using either a Blossom 63matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and alength weight of 2, 3, or 4. In yet another embodiment, the percentidentity between two nucleic acid sequences can be accomplished usingthe GAP program in the GCG software package (Accelrys Inc.), using a gapweight of 50 and a length weight of 3.

Other preferred sequence comparison methods are described herein.

The invention also encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by the polypeptide, e.g., the ability toact as a substrate for an E. coli specific protease. Similarity isdetermined by conserved amino acid substitution. Such substitutions arethose that substitute a given amino acid in a polypeptide by anotheramino acid of like characteristics. Conservative substitutions arelikely to be phenotypically silent. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gln; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247: 1306-1310,1990).

Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, or truncation or a substitution, insertion,inversion, or deletion in a critical residue or critical region, suchcritical regions include the proteolytic cleavage site for aninfection-specific protease.

Amino acids in a polypeptide of the present invention that are essentialfor cleavage by an E. coli specific protease can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science, 244:1081-1085, 1989). The latter procedure introduces a single alaninemutation at each of the residues in the molecule (one mutation permolecule).

The invention also includes polypeptide fragments of the peptidesubstrates or functional variants thereof. The present invention alsoencompasses fragments of the variants of the polypeptides describedherein. Useful fragments include those that retain the ability to act assubstrates for an infection-specific protease.

Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the polypeptide fragment and an additional region fused tothe carboxyl terminus of the fragment.

The biosensors of the present invention can be used in any situationwhere it is desirable to detect the presence or absence of bacteria, andin particular, pathogenic bacteria. For example, bacteria that collectson work surfaces in food manufacturing or food preparation facilities,or health care facilities, and in particular in operating rooms can bedetected with a biosensor as described herein. A substrate, or more thanone substrate, that can be modified by an enzyme secreted by orpresented on the surface of a bacteria is labeled and covalently boundto a collector substrate, such as cotton fibers on the tip of a swab.When more than one substrate is utilized, each may be labeled so as todistinguish it from another (for example, using different detectablelabels) and/or each may be localized in a particular region on the solidsupport. The swab tip is used to wipe the surface suspected of beingcontaminated by bacteria. The swab tip is placed in a medium andincubated using conditions that allow modification of the labeledsubstrate if an enzyme specific for the bound, labeled substrate(s) ispresent.

The present invention also features a kit for detectinginfection-specific bacteria as described herein. The kit can comprise asolid support, for example, having a plurality of wells (e.g., amicrotiter plate), to which a detectably labeled substrate is linked,coupled, or attached. A means for providing one or more buffer solutionsis provided. A negative control and/or a positive control can also beprovided. Suitable controls can easily be derived by one of skill in theart. A sample suspected of being contaminated by a pathogen describedherein is prepared using the buffer solution(s). An aliquot of thesample, negative control, and positive control is placed in its own welland allowed to react. Those wells where modification of the substrate,for example, a color change is observed are determined to contain amicrobial pathogen. Such a kit is particularly useful for detecting aninfection in a subject.

Also encompassed by the present invention is a kit that comprises abiosensor, such as a packaged sterilized wound dressing or a sensor forfood packaging material, and any additional reagents necessary toperform the detection assay.

A method for developing an assay for detecting a pathogenic bacteriathat produces at least one enzyme that is secreted by the cell orpresent on the surface of the cell and a method for using the assay todetect pathogenic bacteria producing the enzyme(s) now follows:

-   -   Step 1) Define an amino acid sequence that uniquely identifies        the prokaryotic microorganism of interest. Alternatively a (one        or more) amino acid sequence that is unique to a specific group        of pathogens, for example, infection-specific pathogens can be        determined.

Select an amino acid sequence, for example, a protein, peptide, orpolypeptide (marker sequence) that uniquely characterizes or marks thepresence of the microorganism or group of microorganisms (for example,infection-specific pathogens) of interest. The selection can beperformed utilizing a bioinfomatic approach, for example, as describedin detail below. One or more amino acid sequences that are unique to aspecific prokaryotic microorganism are determined.

-   -   Step 2) Obtain sufficient enzyme to determine conditions        facilitating optimal modification of a substrate by the enzyme.

Isolate the enzyme from the extracellular medium in which the pathogenicbacteria to be assayed is growing, or from the cell membrane of thebacteria, using standard protein purification techniques, described, forexample, in Ausubel (supra).

Alternatively, if the genetic sequence encoding the enzyme or thelocation of the genetic sequence encoding the enzyme are unknown,isolate and clone the genetic sequence encoding the marker amino acid ofStep 1, or, first determine the genetic sequence, and then proceed asbefore.

-   -   Step 3) Determine the conditions for growth of the prokaryotic        organism and for the production of an enzyme presented on the        surface of the cell or secreted by the cell.

Determine medium required for growth of the specific prokaryoticmicroorganism of interest and for expression of its unique active enzymeinto the medium. Also determine whether a second molecule, for example,an enzyme is required to convert the specific enzyme from an inactiveprecursor form to an active form. To determine if the enzyme has beensecreted in an active form, a sample of the bacterial culture isprovided with chosen potential substrates and cleavage of thesesubstrates is determined. This can be done, for example, by combiningthe bacteria that produce the enzyme with the substrate in theappropriate media and incubating at 37° C. with gentle shaking. Atpreset times (0.1, 0.3, 1.0, 3.0, 5.0, 24 and 48 hours) the samples arecentrifuged to spin down the bacteria, and a small aliquot is removedfor an SDS-PAGE gel sample. After completion of the time course, thesamples are run on a 10-15% gradient SDS-PAGE minigel. Then, theproteins are transferred to Immobilon Pseq (Transfer buffer, 10% CAPS,10% methanol pH 11.0, 15 V for 30 minutes) using a Bio-Rad semi-drytransblotting apparatus. Following transfer of the proteins, the blot isstained with Coomassie blue R-250 (0.25% Coomassie Brilliant Blue R-250,50% methanol, 10% acetic acid) and destained (high destain for 5minutes, 50% methanol, 10% acetic acid; low destain until complete, 10%methanol, 10% acetic acid) followed by sequencing from the N-terminal.Alternatively, the samples can be run on a mass spectrometer in order tomap the sites of proteolytic cleavage using a Voyager Elite Massspectrometer (Perceptive Biosystems, Albertville, Minn.).

-   -   Step 4) Identify any specific substrate(s) of the active enzyme        protease. Examples of potential substrates include proteins,        peptides, polypeptides, lipids, and peptidoglycan subunits.        Label each substrate with a detectable label, for example, a        detectable label described herein, or any other detectable label        known in the art.    -   Step 5) Increase the specificity of the enzyme-substrate        interaction (optional) by determining the active or binding site        of the enzyme (for example, using FRET as described above), then        determining the genetic sequence useful for producing the active        or binding site, and cloning the determined genetic sequence to        generate a more specific substrate.    -   Step 6) Provide a biosensor comprising one or more of the        detectably labeled substrates identified above for detection of        the protease of the pathogenic bacteria of interest.

The substrate can be attached to solid support, for example, a wounddressing, or an article that holds the enzyme and substrate, forexample, a body fluid collection tube or bag, a microplate well, or atest tube. The solid support, if desired, can provide a plurality ofderivatized binding sites for coupling to the substrate, for example,succimidyl ester labeled primary amine sites on derivatized plates(Xenobind plates, Xenopore, Hawthorne, N.J.).

Optionally, unoccupied reactive sites on the solid support are blockedby coupling bovine serum albumin, or the active domain of p26 thereto.p26 is an alpha-crystallin type protein that is used in this case toreduce non-specific protein aggregation. The ability of the p26 proteinto refold heat denatured citrate synthetase before and after coupling tothe surface of the food packaging is used as a control for determiningp26 activity. Alpha-crystallin type proteins were recombinantly producedusing standard recombinant DNA technologies (see Ausubel, supra).Briefly, the plasmid containing the beta sheet-charged core domain ofp26 is electroporated into electrocompetent BL21(DE3) cells (Bio-Rad E.coli pulser). The cells are grown up to an OD₆₀₀ of 0.8, then inducedwith 1 mM IPTG for 4 hours. The cells are spun down, and sonicated inlow buffer (10 mM Tris, pH 8.0, 500 mM NaCl, 50 mM Imidizole) to lyse(Virsonic, Virtis, Gardiner, N.Y.). The lysate is spun down at 13,000×gfor 10 minutes, and the supernatant 0.45 and 0.2 μm filtered. Thisfiltrate is loaded onto a Ni-NTA superose column (Qiagen, Valencia,Calif., cat # 30410). High buffer (10 mM Tris pH 8.0, 500 mM NaCl, 250mM Imidizole) is used to elute the protein.

Allow the enzyme(s) to come into contact with the substrate(s), andmonitor the reaction for a modification in the detectably labeledsubstrate, as described herein. Modification of the substrate indicatesthat the enzyme produced/secreted by the bacteria is present in thereaction. In addition, the absence of modification of the substrateindicates that the enzyme is not present in the sample. If the bacteriaor enzyme is from a wound or other infected area, modification of thesubstrate indicates that the bacteria is present in the wound orinfected area, and that the wound or area is infected, while the absenceof modification of the substrate indicates that the particular bacteriais not present in the wound or area, and that the wound or area is notinfected with that particular bacteria.

EXAMPLES

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

Example 1 Detection of the Presence of E. coli in a Sample

E. coli Assay Development

The Gram-negative bacterium Escherichia coli is the best characterizedhuman pathogen and is known to secrete very few molecules unlessspecifically required for virulence. The virulent strains include thoselikely to cause food poisoning (O157:H7), intestinal disorders (EHECs)or urinary tract infections (UTIs). However, most strains of E. coli canharmlessly coexist with humans and are not likely to cause disease undernormal circumstances.

Although many of the genes are common to other bacteria, E. coli hasdeveloped some unique means of coexistence. A search of the E coli K-12genome by subtraction of several other pathogenic and non-pathogenicbacteria provides a list of genes that are unique to this organism. Thelisting obtained includes the outer membrane proteins phospholipase A,outer membrane protein T (ompT) and several other omp genes.

The gene ompT encodes an enzyme that is found on the outer surface ofthe cell membrane and is used to protect the cell from strongly cationicantimicrobial peptides (defensins) produced by humans. The protein OmpTis a membrane bound protease that has been shown to efficiently cleaveprotamines (salmon milt). The enzyme binds positively charged proteinsand peptides and cleavage occurs preferentially at a site between twopositively charged residues.

The peptide substrates used here were labeled with the fluorescent probeedans (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) and thequencher dye molecule dabcyl ((4-(4-(dimethylamino)phenyl)azo)benzoicacid). The labeled peptides ecot1 (T1) and ecot2 (T2) sequences used areas follows: (SEQ ID NO:1) (T1) Edans - DSRPVRRRRRPRVSK - Dabcyl (SEQ IDNO:2) (T2) Dabcyl - KVSRRRRRGGD - Edans

The bacteria were grown in an incubator overnight at 37° C. in 5 mL BHI(Brain Heart Infusion) media. Each of the resulting cultures was splitinto two samples. One was used as a culture, and the other was spun downby centrifugation and the supernatant was collected. The assays were runin 20 mM tris buffer (pH 7.4) with 150 mM NaCl added. The reaction wascarried out with 5 μL of culture or supernatant and 5 μL of peptidesubstrate (10 mg/mL in water) in 100 μL total volume at 37° C. Thereaction was followed on a fluorimetric plate reader using an excitationwavelength of 355 nm and an emission wavelength of 485 nm.

The first set of experiments was performed by addition of the bacterialculture directly into the assay solution. The protease OmpT is amembrane bound protein and would not be expected to be found secretedinto the media. The first assay to be run used the peptide ecot1 (T1) assubstrate. The results are shown in FIG. 1.

As shown in FIG. 1, protease activity was observed for both E. coli andPseudomonas with the T1 peptide substrate. The same protease assay wasrepeated under identical conditions for substrate ecot2 (T2). Theresults are shown in FIG. 2

As shown in FIG. 2, the sample containing E. coli cells reacted withthis substrate. This peptide appears to be both efficient and selectivefor E. coli.

To test whether the protease is membrane associated, as expected for Ecoli OmpT, the protease assays were repeated with the supernatantsobtained from each bacterial culture. When peptide substrate T1 was usedwith the bacterial culture supernatants, the protease activity observedfor Pseudomonas was still present, but the activity associated with theE. coli cells was not present in the supernatant. This indicates thatthe protease from Pseudomonas is secreted into solution, but the E. coliprotease observed here is membrane bound and may be due to OmpT. Whenpeptide substrate T2 was used with the bacterial culture supernatants,the peptide substrate T2 did not show any reactivity with a secretedprotease from E. coli or any of the other bacteria tested. Thisindicates that peptide T2 appears to be selective for the E. coli outermembrane protease OmpT.

The T2 peptide substrate was further tested for cross reactivity withthe types of conditions and molecules that may be present in a woundenvironment. A fluid that may be present in a wound, at least initially,is serum. In order to test for reactivity with serum the reaction bufferwas modified to by addition 5% fetal bovine serum and the protease assaywas repeated, using the T2 peptide substrate. The results are shown inFIG. 3. As shown in FIG. 3, detection of the presence of E. coli in theE. coli sample occurred in the presence of FBS.

The protease assay was also tested in a simulated wound fluid buffer.The buffer was tris-buffered saline, as described above, to which 5% (byweight) bovine serum albumin was added. The protease assay was repeated,again using the T2 peptide substrate. The results of this assay areshown in FIG. 4. As shown in FIG. 4, the protease reactivity of the E.coli sample was not affected by the simulated wound fluid buffer. Underthese conditions the peptide T2 appears to be a rapid and selectiveprobe for the detection of E. coli cells.

Example 2 Development of Biosensor Surfaces

The attachment of molecules to surfaces can be performed by the use ofseveral different types of interactions. Typically, proteins can beattached to surfaces using hydrophobic, electrostatic, or covalentinteractions. There are many commercially available membranes and resinswith a variety of surface properties. Surfaces can also be chemicallymodified to provide the required surface properties.

Commercially available transfer membranes exist for protein and peptidebinding. They consist of positively and negatively charged polymers suchas ion exchange membrane disc filters and resins. Nitrocellulosemembranes offer hydrophobic and electrostatic interactions. Glass fibermembranes offer a hydrophobic surface that can easily be chemicallymodified to add functional groups. There are also modified polymermembranes that offer reactive functional groups that covalently bindproteins and peptides.

It is also possible to utilize various functional groups on membranes orresins and a crosslinking agent to covalently link to proteins.Crosslinking reagents contain two reactive groups thereby providing ameans of covalently linking two target functional groups. The mostcommon functional groups to target on proteins are amine, thiol,carboxylic acid, and alcohol groups that are used to form intramolecularcrosslinks. Crosslinking agents can be homobifunctional orheterobifunctional and a selection of crosslinking agents of variouslengths are commercially available.

Initially the peptides studied were designed as substrates for bacterialassay development using fluorescence energy transfer (Edans and Dabcyl)for detection. T2, which is selective for E. coli, is an example of sucha substrate, and is described herein.

In order to develop substrates specifically for surface immobilization,several versions of the T2 peptide were made. The peptides were designedto include lysine groups (amine functional group) at one end of thepeptide in the case of T2. The addition of two lysine groups (KK) at oneend of the peptide serve as a “tag” and provide ideal groups forattachment to surfaces through techniques such as electrostaticinteractions or through covalent attachment. The peptide T4 was designedto include a cysteine group (C) and three histidine groups (HHH) at oneend. The addition of a cysteine provides another ideal group or tag toperform covalent attachments through the thiol group. The inclusion ofthree histidine groups also provides the potential for attachment tonickel resins.

The peptide sequence for T2 was modified as shown: (SEQ ID NO: 2) T2(dabcyl-K)VSRRRRRGG(D-edans) (SEQ ID NOS: 3 and 4) T3KKAS(E-edans)VSRRRRRGG(K-dabcyl) (SEQ ID NOS: 5 and 6) T4CHHHAS(E-edans)VSRRRRRGG(K-dabcyl)The pre-peptide tags were added to the original sequences to allow forattachment to a surface.

The protease assay, described herein for detection of E. coli was runwith the modified version of T2, T3. Bacteria (Pseudomonas, E. coli, S.aureus, S. epidermidis, S. salivarius, S. pyogenes, Enterococcus, andSerratia) were grown in an incubator overnight at 37° C. in 5 mL BHI(Brain Heart Infusion) media. The assays were run in 20 mM tris buffer(pH 7.4) with 150 mM NaCl added. The reaction was carried out with 7 μLof culture including cells and media and 3 μL peptide substrate (5 mg/mLin water) in 100 μL total volume at 37° C. The reaction was followed ona fluorimetric plate reader using an excitation wavelength of 355 nm andan emission wavelength of 485 nm. The results are shown in FIG. 5. Asshown in FIG. 5, this assay appears to be specific for E. coli.

Metal chelate (affinity binding) interactions can provide a strongerbond to biological molecules. A his-tag built into the peptidesubstrate, for example T4 can be used to allow linkage to a nickelbinding resin. The resin is incubated with a suitable culture, forexample, E. coli for 30 minutes at 37° C. After centrifugation thebuffer is removed and the pelleted resin is imaged. The fluorescenceproduced by the peptide is then detected. In an example of such adetection assay, E. coli was detected using a biosensor in which ahis-tagged T4 peptide was linked to a nickel binding resin andsubsequently exposed to E. coli cultures or exposed to BHI media withoutbacteria.

Lysine peptide tags, for example, T3 can be used to link to a surfacesuch as UltraBind™ (Pall Gelman Laboratory, Ann Arbor, Mich.). UltraBindis a polyethersulfone membrane that is modified with aldehyde groups forcovalent binding of proteins. Proteins are directly reacted with theUltraBind surface. It is also possible to link proteins or peptides tothe surface using cross linker chains. For example, the carbodiimide,EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride) iscommonly used to link carboxylic acid groups to amines. The linking ofthe peptide with a cross linking agent allows the choice of a linkerchain to extend the peptide off the surface of the membrane while stillcovalently binding it. The linking of the peptide through a cross linkercan be optimized to make the peptide available to the bacterial enzymes.This allows for optimization of the reaction time of the sensor sincepeptide availability is directly related to this parameter.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for detecting the presence or absence of Escherichia coli ina sample, comprising the steps of: a) contacting the sample with adetectably labeled substrate for an enzyme produced and/or secreted byEscherichia coli, under conditions that result in modification of saidsubstrate by said enzyme; and b) detecting the modification or theabsence of the modification of said substrate, wherein modification ofsaid substrate indicates the presence of Escherichia coli in saidsample, and wherein the absence of modification of said substrateindicates the absence of Escherichia coli in said sample.
 2. The methodof claim 1, wherein said enzyme is a protease.
 3. The method of claim 1,wherein said sample is selected from the group consisting of a woundsurface on a subject and a body fluid.
 4. The method of claim 1, whereinsaid substrate is on a solid support.
 5. The method of claim 4, whereinsaid solid support comprises a material required to be free of microbialcontaminants.
 6. The method of claim 4, wherein said solid support is aselected from the group consisting of a wound dressing, a container forholding body fluids, a disk, a scope, a filter, a lens, foam, cloth,paper, a suture, a food packaging material, and a swab.
 7. The method ofclaim 6, wherein said container for holding body fluids is selected fromthe group consisting of a urine collection bag, a blood collection bag,a plasma collection bag, a test tube, a catheter, and a well of amicroplate.
 8. A method for detecting the presence or absence of aninfection in a subject, comprising the steps of: a) contacting a sampleobtained from a subject with a detectably labeled substrate for anenzyme produced and/or secreted by Escherichia coli, under conditionsthat result in modification of said substrate by said enzyme; and b)detecting the modification or the absence of the modification of saidsubstrate, wherein modification of said substrate indicates the presenceof an infection in said subject, and wherein the absence of modificationof said substrate indicates the absence of an infection in said subject.9. The method of claim 8, wherein said enzyme is a protease.
 10. Themethod of claim 8, wherein said sample is a body fluid.
 11. The methodof claim 8, wherein said substrate is on a solid support.
 12. The methodof claim 11, wherein said solid support comprises a material required tobe free of microbial contaminants.
 13. The method of claim 12, whereinsaid solid support is a selected from the group consisting of a wounddressing, a container for holding body fluids, a disk, a scope, afilter, a lens, foam, cloth, paper, a suture, and a swab.
 14. The methodof claim 13, wherein said container for holding body fluids is selectedfrom the group consisting of a urine collection bag, a blood collectionbag, a plasma collection bag, a test tube, a catheter, and a well of amicroplate.
 15. A method for detecting the presence or absence of awound infection in a subject, comprising the steps of: a) contacting awound in a subject with a detectably labeled substrate for an enzymeproduced and/or secreted by Escherichia coli, under conditions thatresult in modification of said substrate by said enzyme; and b)detecting the modification or the absence of the modification of saidsubstrate, wherein modification of said substrate indicates the presenceof a wound infection in said subject, and wherein the absence ofmodification of said substrate indicates the absence of a woundinfection in said subject.
 16. The method of claim 15, wherein saidenzyme is a protease.
 17. The method of claim 15, wherein said substrateis on a solid support.
 18. The method of claim 17, wherein said solidsupport is a wound dressing.
 19. A biosensor for detecting the presenceor absence of Escherichia coli in a sample, said biosensor comprising asolid support and a detectably labeled substrate specific for an enzymeproduced and/or secreted by said microorganism, said substrate attachedto said solid support.
 20. The biosensor of claim 19, wherein the solidsupport comprises a material required to be free of microbialcontaminants.
 21. The biosensor of claim 19, wherein said solid supportis a selected from the group consisting of a wound dressing, a containerfor holding body fluids, a disk, a scope, a filter, a lens, foam, cloth,paper, a suture, a food packaging material, and a swab.
 22. Thebiosensor of claim 21, wherein said container for holding body fluids isselected from the group consisting of a urine collection bag, a bloodcollection bag, a plasma collection bag, a test tube, a catheter, and awell of a microplate.
 23. The biosensor of claim 19, wherein saidbiosensor directly contacts said wound.
 24. A kit for detecting aninfection, comprising a biosensor according to claim 19, and one or morereagents for detecting the enzyme produced and/or secreted byEscherichia coli causing said infection.